1. Field of the Invention
The present invention relates to a focusing method for an examination apparatus, such as a microscope, that can observe fluorescence emitted from a specimen.
2. Description of Related Art
In the related art, fluorescence microscopes are known examination apparatuses. In fluorescence microscopes, excitation light from a light source is focused onto a specimen by an objective lens, fluorescence emitted from the specimen passes through the objective lens and is focused by an imaging lens, and the fluorescence is transmitted through a prism disposed inside a lens barrel to form an image.
In the fluoroscopy carried out with such a fluorescence microscope, the following steps are performed: (a) first, the specimen is initially focused; (b) next, the illumination intensity of the light source is reduced; (c) at the same time, the sensitivity of a photosensor is increased; and (d) the focal position is finely adjusted. While viewing the acquired images, steps (b) and (c) are repeated until a suitable image is displayed, whereupon fluoroscopy can be carried out.
However, many operations are involved in such fluoroscopy, such as performing coarse adjustment and fine adjustment of the focal position while adjusting the illumination intensity. Therefore, this method suffers from the problem that an operator who is unaccustomed to such operations may easily make a mistake.
In addition, recently there have been more applications involving examination of protein-containing specimens (such as GFP). With such protein-containing specimens, however, the fluorescence intensity is very low. Therefore, if the focusing in step (a) described above takes too long or if the illumination intensity for quickly carrying out the focusing in step (a) is increased too much, the level of fluorescence from the specimen decreases due to bleaching of the specimen. This results in a problem in that fluoroscopy cannot be achieved.
On the other hand, scanning laser microscopes have recently been gathering attention. In such microscopes, laser light serving as excitation light is two-dimensionally scanned onto a specimen, fluorescence generated in the specimen passes through a pinhole and is detected in a detector, and the output of the detector is converted to a digital signal. Thereafter, the digital signal is stored in a memory and is displayed as an image. These scanning laser microscopes also have a focus adjusting function for adjusting the focal position of the image.
Japanese Unexamined Patent Application Publication No. HEI-9-218355 discloses an example of a focus adjusting method for a scanning laser microscope. In this method, initially a pinhole is removed from the light path and the focal position is roughly found, based on non-confocal detection, with a photodetector. Then, the pinhole is inserted into the light path and confocal detection is performed with the detector to more precisely find the focal position.
However, in the focusing method in Japanese Unexamined Patent Application Publication No. HEI-9-218355, when using a protein-containing specimen or the like whose fluorescence intensity is extremely low, it also takes a long time to roughly detect the focus position with the non-confocal detection. In order to more quickly perform detection of the focus position with non-confocal detection, the laser beam intensity can be increased; however, if the laser beam intensity is increased too much, there is a problem in that the fluorescence becomes reduced due to bleaching of the specimen, which makes it difficult to perform fluoroscopy.